Chicken gizzard inner lining (GIL) is a by-product of animal meat products. This study examines the chemical characteristics and functional proteins of GIL. Our results demonstrate that GIL is protein-rich and contains essential amino acids for humans. In addition, we analyzed soluble GIL proteins in GIL and categorized the identified proteins into 12 functional groups or processes. Moreover, biochemical analysis revealed that the 18-kDa antrum mucosa protein (Gastrokine-1 protein, GKN-1), which is known to support gastroprotective and physiological activities, was the most abundant protein of GIL. In conclusion, GIL is an important source of proteins and bioactive compounds, such as GKN-1. Therefore, GIL may have some capabilities as important applications in food processing.